In recent years, there has been significant interest in the development of biologic drugs, including peptide biologics (Multard, 2013, Nat. Rev. Drug Discovery 12:329-332; Projan, et al., 2004, Expert Opin. Biol. Therapy 4:1345-1350; Verdine, et al., 2007, Clin. Cancer Res. 13:7264-7270). Peptide biologics have distinct advantages over small molecule drugs. Biologics are typically based on natural bioactive peptides (such as hormones and neuropeptides), and this makes the identification of a “lead compound” much easier than the identification of a small molecule lead compound (Buse, et al., 2009, Lancet 374:39-47; Kreymann, et al., 1987, Lancet 330:1300-1304).
Unfortunately, peptides generally make poor drugs because they undergo rapid proteolysis in vivo, leading to unfavorable pharmacokinetics (Weber, 2004, J. Med. Chern. 47:4135-4141). As a consequence, much of the time in development of peptide biologics is spent on modifying the peptide to reduce proteolysis, while maintaining biological activity (Buse, et al., 2009, Lancet 374:39-47; DeFronzo, et al., 2005, Diabetes Care 28:1092-1100). Some of the strategies reported in the art make use of the incorporation of unnatural amino acids (such as D-amino acids or β-amino acids) into the peptidic chain to overcome proteolysis (Bird, et al., 2010, Proc. Natl. Acad. Sci. 107:14093-14098; Sato, et al., 2006, Curr. Opin. Biotechnol. 27:638-642). Such approaches require identification of appropriate modification sites and are generally time consuming and often met with failure.
Heart disease is two times more common among obese adults and four times more common among diabetic adults, and consistently these conditions affect large numbers of U.S. adults, where 35% of adults are obese and 27% of those 65 and older have diabetes (90% of diabetes cases are Type 2). Both conditions are strongly regulated by peptide hormones and thus amenable to therapeutic intervention. Aside from insulin and glucagon, one of the most well characterized peptides in this class is the gut-derived incretin hormone glucagon-like peptide 1 (GLP-1) (Kreymann, et al., 1987, Lancet 330:1300-1304).
The GLP-1 7-36 residue fragment, which is referred to simply as “GLP-1” hereinafter (SEQ ID NO:1), stimulates insulin and suppresses glucagon secretion, inhibits gastric emptying, and reduces appetite and food intake (Drucker, et al., 2006, Lancet 368:1696-1705). Glucose-stimulated insulin secretion (GSIS) is a phenomenon wherein certain compounds (natural or synthetic) augment the release of insulin from pancreatic β-cell islets in the presence of glucose. These reagents have no effect on insulin secretion in the absence of glucose, and display two advantages over direct stimulators of insulin secretion. First, since they only cause increased insulin secretion in the presence of glucose, they augment the natural physiological mechanism for insulin secretion. Second, compounds that directly stimulate insulin secretion can cause β-cell stress and lead to the death of these vital cells (Maedler, et al., 2005, J. Clin. Endocrinol. Metab. 90:501-506). However, simple treatment by GLP-1 injection is not feasible because it is inactivated through proteolytic cleavage by dipeptidyl peptidase 4 (DPP-4) with a half-life of less than 2 minutes (Kim, et al., 2008, Pharmacol. Rev. 60:470-512). DPP-4 preferentially cleaves after Pro or Ala residues penultimate to the N-terminus and functions as the principal determinant of the circulating half-life for GLP-1 and many other peptides that affect cardiac health (Mentlein, et al., 1993, Eur. J. Biochem. 214:829-835).
Therapeutic approaches for enhancing incretin action include both degradation-resistant GLP-1 receptor (GLP-1R) agonists and inhibitors of DPP-4 activity. Two stabilized incretin mimetics are currently prescribed as injectables taken between once daily and once weekly: exenatide (Byetta®, SEQ ID NO:3) and liraglutide (Victoza®, SEQ ID NO:4). Both induce reductions in fasting, postprandial blood glucose concentrations and hemoglobin Alc (1-2%), which is associated with weight loss (2-5 kg). These incretin mimetics also expand pancreatic 13-cell mass, and have emerged, along with DPP-4 inhibitors such as sitagliptin (Januvia®), as viable treatments for Type 2 diabetes (Drucker, et al., 2006, Lancet 368:1696-1705). While they act along the same hormone signaling axis, the two types of therapies are not mutually exclusive, as DPP-4 inhibitors fail to produce some desirable effects of the peptidomimetics such as appetite suppression and weight loss. Moreover, there is concern that DPP-4 inhibition could increase the risk of cancer (Stulc, et al., 2010, Diabetes Res. Clin. Pract. 88:125-131). DPP-4 exists as both a membrane bound form and a soluble form in circulation due to cleavage of the active site domain from the membrane. Activity of the membrane bound form suppresses non-small cell lung carcinoma cells (Wesley, et al., 2004, Int. J. Cancer 109:855-866). Given that there are concerns about chronic DPP-4 inhibition and some effects are unique to stabilized GLP-1 peptides, there is an interest in using peptides instead of, or in addition to, approved DPP-4 inhibitors.
DPP-4 substrates include not only GLP-1, but also glucose-dependent insulinotropic factor (GIP; SEQ ID NO:5), oxyntomodulin (OXM; SEQ ID NO:6), and brain natriuretic peptide (BNP; SEQ ID NO:7). All of these peptides have half-lives of less than 15 minutes. Similar to GLP-1, the hormones GIP and OXM act as glucose-lowering agents and have been studied extensively as diabetes treatments (Meneilly, et al., 1993, Diabetes Care 16:110-114; Cohen, et al., 2003, J. Clin. Endocrinol. Metab. 88:4696-4701). BNP plays an important role in the body's defense against hypertension and is used as a treatment of congestive heart failure (Del Ry, et al., 2013, Pharmacol. Res. 76:190-198; Grantham, et al., 1997, Am. J. Physiol.—Reg. Int. Comp. Physiol. 272:R1077-R1083). DPP-4 inhibition affects the levels of all of these peptides in circulation. On the other hand, stabilized versions of GIP, OXM, or BNP should act more selectively than DPP-4 inhibition, by impacting only one signaling pathway.
A variety of peptidomimetic strategies have already been applied to stabilizing peptide hormones, including GLP-1. Most of the strategies involve restricting DPP-4 access to the cleavable bond. Exenatide does this using replacement with other natural amino acids. Liraglutide includes a fatty acid modified sidechain, which wraps around the peptide and stabilizes a compact conformation. In the known peptides M1 (SEQ ID NO:8; Deacon, et al., 1998, Diabetologia 41:271-278); M2 (SEQ ID NO:9; Heard, et al., 2013, J. Med. Chem. 56:8339-8351) and M3 (SEQ ID NO:10; Iltz, et al., 2006, Clin. Ther. 28:652-665), access to cleavable bonds is blocked and conformations are stabilized with methyl substitutions. These modifications can extend the half-life for DPP-4 proteolysis, but can compromise GLP-1R affinity (for example, the minimalist M3 has a 6-fold lower affinity; Iltz, et al., 2006, Clin. Ther. 28:652-665). Modifications that increase GLP-1 half-life while sacrificing GLP-1R activation are less effective at achieving the desired effects of regulating glucose and promoting weight loss.
There is a need in the art for straightforward methods of stabilizing peptides, such as peptide hormones or neuropeptides, against proteolytic degradation in vivo. Such methods should allow for the identification of a modified peptide with similar potency to, but increased stability over, the naturally occurring peptide. The present invention addresses this unmet need in the art.